Staurosporine, a widely used protein kinase C inhibitor, completely inhibits both PMA- and insulin-stimulated glucose transport activity in isolated rat adipocytes, possibly at a step that regulates the specific transport activity in isolated rat adipocytes, possibly at a step that regulates the specific translocation of the glucose transporters to the plasma membrane. Human adipose cells are much less responsive to insulin stimulation of glucose transport activity than are rat adipocytes. The intracellular retention process appears to have adapted to release, in the basal state, a greater proportion of th total-cellular pool of GLUT4 to the cell surface of th larger human adipocytes. As a consequence of this adaptation to cell size and surface area, the residual intracellular-reserve pool of GLUT4 that is available to respond to insulin is lower in the human than in the rat adipocyte. We have used a photoaffinity label to quantitate the glucose transporters on the surface of BC3H01 cells. This technique demonstrates that glucose prefeeding 1) decreases the total number of GLUT1 and 2) redistributes the majority of the remaining transporters to an intracellular site where they can now be translocated to the cell surface in response to insulin and PMA. The effects of insulin on the cell surface quantities of GLUT1, GLUT3, and GLUT4 glucose transporters in L6 myotubes were determined. Insulin stimulated the translocation of all three glucose transporter isoforms to the plasma membrane fraction from the light microsome fraction, resulting in equal molar quantities on the cell surface. To test the hypothesis the T3 increases the partitioning of GLUT1 to the cell surface in the liver-derived ARL 15 cell line, we quantitated cell surface GLUT1. We conclude that most of the early (6 h) stimulation of glucose transport by T3 in ARL 15 cells is mediated by an increase in the partitioning of GLUT1 to the plasma membrane. With more chronic T3 treatment (48 h), the enhanced surface partitioning of GLUT1 is persistent, and is superimposed on an increase in total cellular GLUT1, accounting for a further increase in glucose transport. We have observed that LDL receptors localized in intracellular compartments of isolated rat adipose cells seem to be functionally regulated since insulin acutely diminishes the number of receptors by accelerating their rate of degradation. We tested the hypothesis that sulfonylureas promote glucose uptake into 3T3-L1 cells or isolated rat adipocytes through specific high affinity receptors. We conclude that the anti-diabetogenic effects of sulfonylureas are not mediated by a direct action of sulfonylureas to increase uptake into adipose tissue.